Field of the Invention
The present application provides methods of preventing and treating the toxic effects of exposure to organophosphate agents. In embodiments, targeted cationic liposome complexes delivering nucleic acid molecules encoding butyrylcholinesterase and nucleic acid molecules encoding a polyproline rich sequence are administered. Suitably, the administration is via inhalation. Also provided are cationic liposome complexes and methods of making the complexes for such administration.
Background of the Invention
Organophosphate agents (OP) are commonly used as pesticides, insecticides and drugs for treatment of medical conditions such as glaucoma and Alzheimer's disease. Unfortunately, they have also been developed for use as nerve agents such as sarin, soman, VX, and tabun. These OP compounds are among the most toxic chemicals known. Exposure to even small amounts can be fatal. Death occurs from asphyxiation resulting from paralysis of the diaphragm and intracostal muscles, depression of the brain respiratory center, bronchospasm, convulsions and excessive salivation (reviewed in 1). The mechanism of OP poisoning is the phosphorylation of the serine hydroxyl group in the active site of Acetylcholinesterase (AchE) which leads to irreversible inactivation of the enzyme. AchE, which hydrolyzes Acetylcholine (Ach) at the synaptic space, is an essential enzyme in neurotransmission. Inactivation of AchE results in a rapid buildup of Ach subsequently producing PNS cholinergic hyperstimulation and death. In the CNS, cholinergic hyperexcitability increases neuronal firing triggering convulsions and acute neuronal cell death.
Although there are antidotal treatments for post-exposure use, they have shown limited efficacy, produce serious side effects, and do not prevent incapacitation (transient or permanent) or irreversible brain damage (1,2). Thus, prophylactic measures are being sought. One approach for counteracting OP toxicity is the use of a bioscavenger to sequester and neutralize these compounds. Of the bioscavengers tested, human serum butyrylcholinesterase (BChE) (also called pseudocholinesterase, or cholinesterase) appears to be the most suited for human use (3). BChE is a serine enzyme present in almost every tissue including plasma, brain, muscle, kidney, liver and lung (4). Human BChE (hBChE) (340 kDa) in serum is a globular tretrameric molecule with a T1/2 of 11-14 days and is composed of four identical subunits and is protected from proteolysis through heavy glycosylation (5). The assembly of the individual subunits into the tetramer requires the presence of a polyproline rich peptide derived from lamellipodin (5) or from rat collagen tail (AChE Q subunit) (Bon paper, Krejci paper, Antamirano paper), or any other polyproline rich protein. BChE is naturally expressed at relatively high levels, 4 times that of the average gene (4). It also plays an important role in the degradation of numerous ester-containing drugs and is a natural bioscavenger of cholinesterase inhibitors, including potent OP nerve agents.
Each molecule of hBChE neutralizes one molecule of OP (6). It has been reported that pretreatment with recombinant human BChE and human serum BChE could protect animals (including rodents, guinea pigs, pigs and non-human primates) from up to 5 times LD50 of nerve agents (6,7). The irreversible binding and inactivating function of BChE with a broad spectrum of OP poisons make it an ideal candidate for a prophylactic treatment against nerve agents. In addition to its use for a variety of wartime pre- and post-exposure scenarios, it also has potential use as a pretreatment for first responders reacting to intentional/accidental nerve gas release and as a post-exposure therapy for pesticide overexposure, cocaine overdose, or succinyl-choline-induced apnea (s).
It has been estimated that in a human, a BChE dose of 250 mg/70 kg is required to achieve efficient protection following a challenge with one LD50 of OPs (3). However, the naturally occurring amount of this bioscavenger enzyme in blood (˜8-72 mg/6 L) is too low to achieve adequate protection due to the stoichometric and irreversible binding of; and the interaction between, the OP and BChE; the unfavorable OP/BChE mass ratio; and the aging of the enzyme (4,9). Thus, it is critical to develop a means to significantly increase the level of BChE expression and amount in plasma. Toward this end, different strategies are being developed. The most straightforward is the direct injection of a large dose of highly purified natural hBChE to increase the amount in the bloodstream. This has proven to be successful for protection against lethal doses of soman and VX but, is not practical for battlefield use. Moreover, use of transgenic animals and cell culture has not been able to produce sufficient quantities of hBChE to be practical and feasible for use.
Derivation of BChE mutants capable of reactivating spontaneously (making them available to bind and deactivate additional molecules of OP) is another approach being employed with some success. BChE was shown to gain OP hydrolase activity and increased reactivation when a Histidine was substituted for Glycine at position 117 (G117H) (10,11). This mutant is efficient at hydrolyzing the acetylcholinesterase inhibitor echothiophate and can also efficiently hydrolyze the nerve agents sarin and VX (9). More importantly, transgenic mice expressing the G117H mutant are resistant to OP (12). Although upwards of 60 BChE mutants have been produced, the G117H remains one of the most efficient and studied to date (9). However, as yet, no means of efficiently delivering or producing for extended periods of time in the body after administration, a BChE or tetrameric form of active mtBChE via non-invasive routes, has been developed.
There is, therefore, an urgent need to develop technologies and methods to deliver BChE for prevention and treatment of exposure to OP agents. The present invention fulfills these needs by providing cationic-liposome-based drug delivery systems for such treatment and/or prevention.